Articles

Single-dose versus 7-day-dose metronidazole for the treatment of trichomoniasis in women: an open-label, randomised controlled trial

Patricia Kissinger, Christina A Muzny, Leandro A Mena, Rebecca A Lillis, Jane R Schwebke, Laura Beauchamps, Stephanie N Taylor, Norine Schmidt, Leann Myers, Peter Augostini, William E Secor, Martina Bradic, Jane M Carlton, David H Martin

Summary Background Among women, trichomoniasis is the most common non-viral sexually transmitted infection worldwide, Lancet Infect Dis 2018; and is associated with serious reproductive morbidity, poor birth outcomes, and amplified HIV transmission. Single- 18: 1251–59 dose metronidazole is the first-line treatment for trichomoniasis. However, bacterial vaginosis can alter treatment Published Online efficacy in HIV-infected women, and single-dose metronidazole treatment might not always clear infection. We October 5, 2018 http://dx.doi.org/10.1016/ compared single-dose metronidazole with a 7-day dose for the treatment of trichomoniasis among HIV-uninfected, S1473-3099(18)30423-7 non-pregnant women and tested whether efficacy was modified by bacterial vaginosis. See Comment page 1169 Department of Epidemiology Methods In this multicentre, open-label, randomised controlled trial, participants were recruited at three sexual (Prof P Kissinger PhD, health clinics in the USA. We included women positive for Trichomonas vaginalis infection according to clinical Prof D H Martin MD, screening. Participants were randomly assigned (1:1) to receive either a single dose of 2 g of metronidazole (single-dose N Schmidt MPH) and Department of Global group) or 500 mg of metronidazole twice daily for 7 days (7-day-dose group). The randomisation was done by blocks Biostatistics and Data Science of four or six for each site. Patients and investigators were aware of treatment assignment. The primary outcome was (Prof L Myers PhD), Tulane T vaginalis infection by intention to treat, at test-of-cure 4 weeks after completion of treatment. The analysis of the University School of Public primary outcome per nucleic acid amplification test or culture was also stratified by bacterial vaginosis status. This trial Health and Tropical Medicine, Tulane University, is registered with ClinicalTrials.gov, number NCT01018095, and with the US Food and Drug Administration, number New Orleans, LA, USA; Division IND118276, and is closed to accrual. of Infectious Diseases, University of Alabama at Findings Participants were recruited from Oct 6, 2014, to April 26, 2017. Of the 1028 patients assessed for eligibility, Birmingham, Birmingham, AL, USA (C A Muzny MD, 623 women were randomly assigned to treatment groups (311 women in the single-dose group and 312 women in the Prof J R Schwebke MD); Division 7-day-dose group; intention-to-treat population). Although planned enrolment had been 1664 women, the study was of Infectious Diseases stopped early because of funding limitations. Patients in the 7-day-dose group were less likely to be T vaginalis positive (Prof L A Mena MD, at test-of-cure than those in the single-dose group (34 [11%] of 312 vs 58 [19%] of 311, relative risk 0·55, 95% CI L Beauchamps MD) and Department of Population 0·34–0·70; p<0·0001). Bacterial vaginosis status had no significant effect on relative risk (p=0·17). Self-reported Health Science (Prof L A Mena), adherence was 96% in the 7-day-dose group and 99% in the single-dose group. Side-effects were similar by group; the University of Mississippi most common side-effect was nausea (124 [23%]), followed by headache (38 [7%]) and vomiting (19 [4%]). Medical Center, University of Mississippi, Jackson, MS, USA; Section of Infectious Diseases, Interpretation The 7-day-dose metronidazole should be the preferred treatment for trichomoniasis among women. Louisiana State University Health Sciences Center, Funding National Institutes of Health. New Orleans, LA, USA (R A Lillis MD, Prof S N Taylor MD, Prof D H Martin); Division of Copyright © 2018 Elsevier Ltd. All rights reserved. Parasitic Diseases and Malaria, Centers for Disease Control and Introduction and preterm delivery) in women.4 Several studies have Prevention, Atlanta, GA, USA 5 (W E Secor PhD, P Augostini); Among women, trichomoniasis is estimated to be the found that trichomoniasis co-occurs with other STIs and and Center for Genomics and 6 most common non-viral sexually transmitted infection amplifies HIV acquisition. Systems Biology, Department (STI) worldwide, and is as prevalent as chlamydia, Both WHO and the US Centers for Disease Control of Biology, New York gonorrhoea, and syphilis combined.1 Trichomoniasis is and Prevention (CDC) recommend a single 2 g dose of University, New York, NY, USA (Prof J M Carlton PhD, not a reportable disease, but an estimated 143 million oral metronidazole or tinidazole as first-line treatment M Bradic PhD) new cases occur globally each year in women aged and a 7-day dose of oral metronidazole (400 mg or 500 mg Correspondence to: 1 15–29 years. The prevalence of trichomoniasis has been twice daily for 7 days) as second-line treatment for Prof Patricia Kissinger, estimated to be around 5% among women worldwide1 Trichomonas vaginalis infections.7,8 In most settings, Department of Epidemiology, and around 1·8% among women in the USA, but nearly metronidazole is used more often than tinidazole, Tulane University School of five times higher among African American women.2 because purchase costs are lower.9 Public Health and Tropical Medicine, Tulane University, Trichomoniasis has been associated with serious Evidence has shown that the single-dose treatment of New Orleans, LA 70112, USA reproductive morbidity (eg, vaginitis, cervicitis, ure-​ metronidazole might be insufficient to treat tricho­moniasis. [email protected] thritis, and endometritis)3 and poor birth outcomes A meta-analysis10 of six published studies found that women (eg, premature rupture of membranes, low birthweight, who received the 7-day-dose metronidazole had 46% fewer www.thelancet.com/infection Vol 18 November 2018 1251 Articles

Research in context Evidence before this study was mostly found among women with co-occurring bacterial Among women, trichomoniasis is the most common non-viral vaginosis. The purpose of this study was to re-evaluate the sexually transmitted infection worldwide. It has been associated efficacy of single-dose metronidazole compared with 7-day-dose with increased reproductive and perinatal morbidity, and metronidazole among HIV-uninfected women, and to amplified HIV acquisition. A single 2 g dose of oral metronidazole examine treatment differences by bacterial vaginosis status. is the recommended first-line treatment of trichomoniasis, with Added value of this study a 7-day dose (400 or 500 mg twice daily for 7 days) as Our findings add to the evidence that 7-day-dose second-line treatment. A meta-analysis of six published studies metronidazole is superior to single-dose metronidazole for found that multiple doses of metronidazole resulted in fewer the treatment of trichomoniasis in women, irrespective of treatment failures than single-dose treatment. However, five of bacterial vaginosis status. the studies were done more than 30 years ago. In our 2010 study with HIV-infected women, we found that women receiving the Implications of all the available evidence 7-day-dose metronidazole treatment were half as likely to be The 7-day dose should be the preferred first-line treatment for Trichomonas vaginalis-positive at test-of-cure compared with T vaginalis infection. Treatment guidelines for trichomoniasis in those receiving the single-dose treatment, but this difference HIV-uninfected women should be refined accordingly.

treatment failures than did women who received the single and accommodate for the possibility of lack of adherence dose, and the proportion of patients with positive test-of- and sexual re-exposure among women who received cure (TOC) after single-dose metronidazole ranged from treatment over multiple days. 6·2% to 18%. Five of the six studies concluded that single- Women who attended one STI clinic in each of three US dose treatment was similar to the 7-day treatment.11–15 cities (Birmingham, AL; Jackson, MS; and New Orleans, However, all but one of these studies were done over LA) for clinical screenings and who had a T vaginalis 30 years ago, when clinical trial protocols were not as infection were referred to study staff by their clinical rigorous as today.16 Most of the studies were not adequately providers. powered to detect meaningful differences, and all were Inclusion criteria for participants were female sex, done before the availability of nucleic acid amplification English speaking, reached the age of majority (between tests (NAATs), which are three-to-five times more sensitive 18 and 19 years, depending on site requirements), than microscopy for the diagnosis of trichomoniasis.17–21 The positive for T vaginalis infection according to clinical most recent study that was included in the meta-analysis screening (microscopy or NAAT) confirmed by at least was our 2010 multicentre study of trichomoniasis one study test (NAAT or culture), willingness to refrain treatment.22 We used newer clinical trial methods but only from all alcohol use during treatment and for 24 h after included HIV-infected women with laboratory-diagnosed treatment, willingness to be treated with metronidazole, T vaginalis. We found that women receiving the 7-day-dose and willingness to be randomly assigned to either of the metronidazole treatment were half as likely to be T vaginalis- study groups. positive at TOC compared with those receiving the single- The exclusion criteria were HIV infection, pregnancy dose treatment.22 Subsequent analysis, however, found or breastfeeding, previous enrolment in this study, superiority in the 7-day-dose group only among the patients incarceration, any medical contraindications to metro-​ who had bacterial vaginosis according to the Nugent score, nidazole (eg, currently taking disulfiram, phenytoin, or suggesting that altered vaginal flora could have interfered warfarin), a history of alcoholism or known liver damage, with the single-dose treatment.23 treatment with any medication used to treat tricho- The purpose of this study was to re-evaluate the efficacy moniasis or bacterial vaginosis (including metronidazole, of single-dose metronidazole compared with 7-day-dose tinidazole, secnidazole, acetarsol, boric acid, furazolidone, metronidazole among HIV-uninfected women, using a and paromomycin) within the previous 14 days, inability or sample size with sufficient power and advanced unwillingness to provide informed consent, unwillingness T vaginalis diagnostics. A secondary aim was to examine to be randomised, and inability or unwillingness to return treatment differences by bacterial vaginosis status. for a follow-up visit 4 weeks after completion of treatment. The study received ethics approval from the institutional Methods review boards of Tulane University, Louisiana State Study design and participants University Health Sciences Center (LSUHSC), University This was a multicentre, open-label, randomised con-​ of Alabama at Birmingham, University of Mississippi trolled trial comparing the efficacy of 7-day-dose Medical Center, Jefferson County Department of Health, metronidazole treatment with the standard single-dose and the Mississippi State Department of Health. An metronidazole treatment of trichomoniasis. An open- independent Data Safety and Monitoring Board monitored label design was used to simulate real-world conditions the data every 6 months, with a priori stopping rules. The

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study was stopped early because of funding limitations and transcription-mediated amplification and hybridisation never reached half of the anticipated sample size, so an protection assay technologies. At the time of the study, interim analysis could not be done. Because of the this assay was allowed for investigational use only, and sensitive nature of the surveys both at enrolment and was performed by use of the direct tube sampling system. TOC, a Certificate of Confidentiality was also obtained The assay is now available for clinical use.26 All tests were from the US Department of Health and Human Services. done at the LSUHSC laboratory (New Orleans, LA, USA), Women who discovered they were pregnant while taking according to the manufacturer’s instructions. the medication or who had any serious adverse events For T vaginalis culture, self-collected vaginal swabs were followed up according to the institutional review were placed into the T vaginalis culture InPouch medium board’s protocol for each site. (Biomed Diagnostics, White City, OR, USA), incubated at Study enrolment was open from Oct 6, 2014, to 37°C and read following the manufacturer’s protocol by April 26, 2017. The study was completed on June 5, 2017. trained personnel. Three readings were done over 7 days. After eligibility screening and written informed consent, The cultures were considered T vaginalis-positive if any the participants were asked to take a survey, provide live trichomonads were detected, and T vaginalis-negative urine for pregnancy testing, and self-collect vaginal after three negative pouch readings.27 swabs for T vaginalis culture, NAAT, and Gram stains. Gram stains for Nugent score determination were read by an experienced technician at the LSUHSC Randomisation and masking laboratory. Samples were periodically evaluated for quality Participants were randomly assigned (1:1) to receive assurance following Clinical Laboratory Improvement either 500 mg of metronidazole twice daily for 7 days Amendments by the US Food and Drug Administration (7-day-dose group) or the standard single dose of 2 g of (FDA).28 Women were considered to have bacterial metronidazole (single-dose group). Group assignment vaginosis if they had a Nugent score of 7 or higher.29 was done with sealed, sequentially numbered envelopes Participants in the single-dose group were treated with that contained the randomly chosen treatment group. 2 g of oral metronidazole (four pills of 500 mg each) in a These envelopes were prepared before the start of the single dose. Participants in the 7-day-dose group were study by a non-investigator who used SAS software to treated with 500 mg oral metronidazole (one pill) taken develop a randomisation scheme of blocks of four or twice daily for 7 days. Participants in both groups were six for each site. A list containing the envelope number asked to take the first dose at the clinic under direct and allocation group was kept in an electronic file that observation by study staff and were offered crackers or was not accessed until the end of the study. Envelopes cookies to prevent nausea. For the participants in the were kept at each site, and study staff pulled envelopes treatment group, the remaining pills were dispensed in a sequentially and documented the treatment group, container with a child-proof cap. envelope number, and lot number of the treatment Participants in both groups were asked to tell all received. All patients, clinicians, and study staff were their sexual partners of their exposure to T vaginalis aware of the allocation, but the treatment group was and to encourage them to seek treatment. Because of masked from all laboratory technicians. legal or institutional restrictions, none of the participating clinics routinely provided expedited partner treatment Procedures (presumptive treatment for trichomoniasis given to The surveys at baseline and at the TOC appointment partners without a clinic visit) but some were given (4 weeks after completion of treatment) were done with expedited partner treatment at the clinic’s discretion. an audio computer-assisted self-administered interview All participants received the standardised advice by (ACASI) software to collect demographics, substance use, study personnel to refrain from unprotected sexual vaginal hygiene practices, contraception use, and sexual intercourse until symptoms had resolved, and until they behaviour data for each participant. Symptoms commonly and their partners had completed the treatment regimen. reported with trichomoniasis (unusual vaginal discharge, Participants were also advised to refrain from alcohol vaginal odour, vaginal itching or irritation, painful consumption while taking the medication and for 24 h urination, pelvic pain)24 were systematically elicited by the after completion. Participants were informed of the ACASI. The survey at the TOC appointment was similar possibility of adverse events related to treatment with to the survey at baseline, but it had additional questions metronidazole, and of the possibility of a change in taste about sexual exposure, treatment adherence, partner sensation and discolouration of urine. Participants in the treatment, symptoms during follow-up, and side-effects treatment group were also advised on the importance of of the medication (including nausea, vomiting, and taking all doses of the medication. headaches). The survey was modelled after surveys from A TOC appointment was scheduled 4 weeks after our previous studies.22,25 completion of treatment, with an appointment window of For T vaginalis NAAT, self-collected vaginal swabs were 3–12 weeks. Women were not screened before the 3-week tested for T vaginalis with the Aptima Trichomonas TOC, because of the potential for false-positive NAAT vaginalis Assay (Hologic, Bedford, MA, USA), which uses results from remnant T vaginalis DNA.30,31 Participants www.thelancet.com/infection Vol 18 November 2018 1253 Articles

observed only in women with bacterial vaginosis. 1028 participants assessed for eligibility Stratification by bacterial vaginosis status was the secondary analysis of the primary outcome. The secondary outcomes were to find the probable 405 excluded 186 ineligible cause of a T vaginalis-positive result at TOC using the 38 refused to be randomised genotyping results and sexual histories, and to find out if 20 pregnant or breastfeeding 15 lost to follow-up most of the infections are due to treatment failure, re- 13 underage exposure, sexual exposure to a new partner, or reduced 9 did not give consent 6 treated for bacterial vaginosis in the susceptibility of T vaginalis to metronidazole. Results for past 14 days the secondary outcomes will be presented in a separate 6 HIV positive report. 79 other (previously enrolled, unable to take medication, do not speak English) 219 declined to enrol Statistical analysis 123 had no time 58 not interested We calculated that a sample size of 1664 participants 23 too upset about their diagnosis would provide statistical power of 80%. Our calculations 15 other were based on inputs from our unpublished pilot study with HIV-uninfected women, and from our previous

623 randomised study of trichomoniasis treatment in HIV-infected women.22 We assumed a T vaginalis-positive TOC rate of 16·8% for single-dose treatment and 10·7% for 7-day-dose treatment, inflated the sample size by 15% for the 312 assigned to 7-day-dose metronidazole group 311 assigned to single-dose metronidazole group potential of interclass correlation between the three sites, and inflated the sample size by a further 20% to account for the potential loss to follow-up. 42 discontinued treatment 41 discontinued treatment 10 post-randomisation 8 post-randomisation Study data were collected and managed with REDCap exclusions exclusions electronic data capture tools (version 7.4) hosted at Tulane 2 withdrew 33 lost to follow-up 1 30 lost to follow-up University (New Orleans, LA, USA). REDCap is a secure, web-based application designed to support data capture for research studies. It provides an intuitive interface for 270 completed treatment 270 completed treatment validated data entry, audit trails for tracking of data manipulation and export procedures, automated export procedures for seamless data downloads to common 312 included in intention-to-treat analysis 311 included in intention-to-treat analysis statistical packages, and procedures to import data from 270 included in per-protocol analysis 270 included in per-protocol analysis external sources. We measured the baseline characteristics associated Figure: Trial profile with trichomonas infection.2,32 The primary outcome analysis of T vaginalis infection at TOC was by intention to treat. Missing outcome data were imputed 20 times for provided their contact information and were given an participants who were lost to follow-up, using the fully appointment card and compensation worth US$20. conditional method in SAS (version 9.4) PROC MI. Participants were reminded of their TOC visit by use of Because the sites did not enrol the same number of their preferred method of contact. At the TOC participants, we had to account for differences between appointment, data collection included a survey and self- sites.33 Generalised estimating equation (GEE) regression collected vaginal swabs for NAAT, culture, and Gram methods were used, including an exchangeable corre-​ stain; participants who completed the TOC visit received lation matrix and clustering by site. Rate differences and compensation worth $50. 95% CIs, as well as GEE-derived relative risks and 95% CIs, were calculated. Intention-to-treat analyses for Outcomes all randomised participants were stratified by baseline The primary outcome was a T vaginalis-positive result at bacterial vaginosis status. the TOC appointment (ie, 4 weeks after completion of Four sensitivity analyses of the primary outcome were treatment), with NAAT or culture. NAATs were centrally also done. We redid all analyses by reclassifying all assessed, but the cultures were done at the laboratory of missing results as negative at TOC in the intention-to- each study site. Allocation was masked from technicians treat population, by reclassifying all missing results as at all sites. The trial hypothesis was that women receiving positive at TOC in the intention-to-treat population, by the 7-day-dose metronidazole would be less likely to have using the T vaginalis culture results as the outcome a T vaginalis-positive TOC than women receiving single- (to remove the possibility of false positives by T vaginalis dose metronidazole, and that this effect would be NAAT) in the per-protocol population and by using

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NAAT and T vaginalis culture results as the outcome in 7-day-dose Single-dose the per-protocol population. metronidazole group metronidazole group Adverse events and serious adverse events were Demographics compared by treatment group. Statistical analyses were Age ≥30 years 116/310 (37%) 132/309 (43%) done either with the χ² test or Fisher’s exact test. All African American 298/310 (96%) 293/309 (95%) analyses were done using SAS version 9.4, under the Study site supervision of the team biostatistician. Jackson, MS 77/312 (25%) 78/311 (25%) The trial is registered with ClinicalTrials.gov, number New Orleans, LA 34/312 (11%) 34/311 (11%) NCT01018095, and with the FDA, number IND118276. Birmingham, AL 201/312 (64%) 199/311 (64%) Role of the funding source Substance use behaviour The funder of the study had no role in study design, data Binge drinking in past week 57/310 (18%) 56/309 (18%) collection, data analysis, data interpretation, or writing of Smoking 145/310 (47%) 142/308 (46%) the report. The corresponding author had full access to Sexual behaviours all the data in the study and had final responsibility for More than one male sexual partner 119/310 (38%) 122/309 (40%) the decision to submit for publication. Any number of female partners 27/310 (9%) 30/309 (10%) Vaginal douching at least once a month 117/309 (38%) 114/309 (37%) Results Use of hormonal contraception 60/310 (19%) 58/309 (19%) Participants were recruited from Oct 6, 2014, to Taking the 7-day dose will be difficult or somewhat 91/310 (29%) 96/309 (31%) difficult April 26, 2017. The study was completed on June 5, 2017. Of the 1028 women with T vaginalis infections that were Clinical factors approached, 623 enrolled in the study (figure). The study Bacterial vaginosis per Nugent score 146/306 (48%) 148/303 (49%) was stopped early because of funding limitations, and we Trichomoniasis in past year per self-report 53/309 (17%) 54/307 (18%) enrolled 623 (37%) of the 1664 patients that were planned Bacterial vaginosis in past year per self-report 48/310 (15%) 65/308 (21%) for our intended sample size. Of the 623 women enrolled Yeast infection in past year per self-report 87/301 (29%) 76/297 (26%) in the study, 312 women were randomly assigned to the Symptoms of trichomoniasis 248/311 (80%) 248/311 (80%) 7-day-dose group and 311 to the single-dose group. All Expedited partner treatment 21/307 (7%) 27/309 (9%) women received the dose allocated to them and were Table 1: Baseline characteristics of the intention-to-treat population included in the intention-to-treat population. Baseline characteristics were well balanced between groups (table 1). Most women who enrolled were African 155 in Jackson, MS; 13 [19%] of 68 in New Orleans, LA; American (591 [96%] of 619) and the median age was 51 [13%] of 400 in Birmingham, AL; p=0·33). Sexual 27 years (range 18–64). The study site in Birmingham, exposure (either to baseline or a new partner) during AL, recruited 400 (64%) of 623 participants. At baseline, follow-up did not differ significantly between treatment 294 (48%) of 609 women had bacterial vaginosis per groups (99 [37%] of 270 in the 7-day-dose group, 89 [33%] of Nugent criteria, and 231 (37%) of 618 did vaginal 270 in the single-dose group, p=0·37). douching at least once per month. Participants had the Adherence to treatment was lower in the 7-day-dose option of not answering any question; this accounts for group than in the single-dose group (253 [96%] of 265 vs variations in the denominators for some of the baseline 264 [99%] of 266, p=0·006). Of the 603 women who variables. reported partners at baseline, 43 (7%) received expedited Participants were screened for T vaginalis via NAAT partner treatment (2 g single-dose metronidazole) for and culture to confirm their clinical screening. In the their sexual partners, with no substantial difference in 7-day-dose group, 239 (77%) of 312 were tested by NAAT provision of treatment by group (table 1). and culture, 16 (5%) were tested by NAAT only, and Overall, 80 (15%) of 540 patients were positive at TOC. 57 (18%) were tested by culture only. In the single-dose One patient was assessed by culture only, 17 by NAAT group, 237 (76%) of 311 were tested by NAAT and culture, only, and 62 by both NAAT and culture. In the intention- 14 (5%) were tested by NAAT only, and 60 (19%) were to-treat analysis (with data imputed for participants who tested by culture only. were lost to follow-up), the proportion of women who Of the 623 patients included in the study, were positive at TOC was lower in the 7-day-dose group 540 (87%) returned for their follow-up visit. The mean than in the single-dose group (34 [11%] of 312 women vs time from completion of treatment to TOC visit was 58 [19%] of 311 women, p=0·0008, relative risk [RR] 0·55, 4·9 weeks (SD 1·6) for the study sample, and did not differ 95% CI 0·34–0·70). When bacterial vaginosis was present, between groups (p=0·60). The proportion of patients who women in the 7-day-dose group were less likely to have a did not return for follow-up did not differ significantly positive result at TOC than women in the single-dose between treatment groups (42 [14%] of 312 patients in the group (p=0·0002, RR 0·59, 0·43–0·80). In the absence of 7-day-dose group vs 41 [13%] of 311 patients in the single- bacterial vaginosis, women receiving the 7-day dose were dose group, p=0·92) or between study sites (19 [12%] of also less likely to have a positive result at TOC compared www.thelancet.com/infection Vol 18 November 2018 1255 Articles

7-day-dose Single-dose 7-day-dose vs single-dose Relative risk (95% CI) p value* metronidazole metronidazole difference (95% CI) group group

Primary outcome analyses by intention to treat† Trichomonas vaginalis infection at test-of-cure 34/312 (11%) 58/311 (19%) –7·8 (–2·2 to –13·3) 0·55 (0·34 to –0·70) <0·0001 Among patients with bacterial vaginosis at baseline 16/125 (13%) 26/125 (21%) –8·0 (–12·8 to –20·8) 0·59 (0·43 to 0·80) 0·0002 Among patients without bacterial vaginosis at 13/139 (9%) 24/140 (17%) –7·8 (–0·2 to –15·8) 0·57 (0·45 to 0·71) <0·0001 baseline Sensitivity analyses of primary outcome All missing TOC results reclassified as negative 29/312 (9%) 51/311 (16%) –7·1 (–1·9 to –12·4) 0·57 (0·45 to 0·71) <0·0001 All missing TOC results reclassified as positive 71/312 (23%) 92/311 (30%) –6·8 (–0·1 to –13·7) 0·77 (0·70 to 0·85) <0·0001 T vaginalis culture results as outcome‡ 22/269 (8%) 41/270 (15%) –7·0 (-1·3 to –12·7) 0·54 (0·39 to 0·75) 0·0002 NAAT and T vaginalis culture results as outcome‡ 29/270 (11%) 51/270 (19%) –8·2 (–2·2 to –14·1) 0·57 (0·45 to 0·71) 0·008

TOC=test-of-cure. NAAT=nucleic acid amplification test. *Relative risks and p values were derived from generalised estimating equation (GEE) analysis. †Missing data imputed using the fully conditional method in SAS. ‡In the per-protocol population.

Table 2: Primary outcome and sensitivity analyses

Of those patients who returned for the TOC, 179 (33%) 7-day-dose Single-dose p value metronidazole metronidazole reported a treatment-related side-effect (table 3). The group (n=270) group (n=270) most common was nausea (23%), followed by headache Any adverse event during 89 (33%) 90 (33%) 0·90 (7%) and vomiting (4%). The proportion of patients with follow-up these events did not differ significantly by study group. Adverse events Two spontaneous abortions were reported among Nausea 63 (23%) 61 (23%) 0·84 women who tested negative for pregnancy at baseline Vomiting 13 (5%) 6 (2%) 0·10 and discovered they were pregnant during follow-up. Headache 23 (9%) 15 (6%) 0·18 Both were in the 7-day-dose group. Dizziness 6 (2%) 2 (1%) 0·15 Bad or metallic taste in 4 (2%) 3 (1%) 0·70 Discussion mouth Trichomoniasis is highly prevalent worldwide and has Vaginal itching 3 (1%) 1 (<1%) 0·32* been associated with increased reproductive and Fatigue 2 (1%) 0 0·32* perinatal morbidity,34 and amplified HIV acquisition.1,6 In Rash 2 (1%) 0 0·32* this randomised trial, treatment of trichomoniasis with Other side-effects† 9 (4%) 9 (3%) 1·00 7-day-dose oral metronidazole (500 mg twice daily for Serious adverse events 7 days) resulted in 45% fewer positive results after Spontaneous abortion‡ 0 2 (1%) 0·32* 4 weeks than single-dose oral metronidazole (2 g single dose) among HIV-uninfected women. These findings are *Adverse events were compared with Fisher’s exact test. †Yeast infection, consistent with our 2010 multicentre study22 of abdominal pain, numbness, dysuria, loss of appetite, altered mental status, myalgia, generalised itching, lightheadedness, restlessness, verbal intimidation by trichomoniasis treatment with HIV-infected women, and partner, and stiff neck. ‡Two women tested negative at baseline and discovered a previous meta-analysis10 of six published studies of they were pregnant during follow-up. trichomoniasis treatment. Table 3: Adverse events and serious adverse events We did not find treatment differences according to bacterial vaginosis status, by contrast with our previous study with HIV-infected women.23 The reason for this with women receiving the single dose (RR 0·57, 0·45–0·71, result is not entirely clear. Although the validity of a p<0·0001). Bacterial vaginosis status had no significant Nugent score can be affected by the technician’s 29 effect on relative risk (pinteraction=0·17). expertise, the same technician read the Gram stains in When all missing results were classified as negative, the both studies, so this variability is not a likely factor. women in the 7-day-dose group had fewer positives at TOC One possible explanation of this result is that host factors than did those in the single-dose group (table 2). Findings affected treatment efficacy; HIV status seems to affect were similar when all missing results were classified as the vaginal microbiota with and without presence of positive. When T vaginalis culture was used as the outcome, bacterial vaginosis.35 Future studies should examine how women receiving the 7-day dose were less likely to be the vaginal microbiota affects the treatment of tricho­ positive at TOC than women receiving the single dose. Per- moniasis. protocol analysis of complete cases showed that women in Concomitant bacterial vaginosis is common among the 7-day-dose group had fewer positive T vaginalis results women with trichomoniasis.36 48% of the women in this than those receiving the single dose. study had bacterial vaginosis per Nugent score, yet

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bacterial vaginosis had not been diagnosed by the Single-dose treatment for trichomoniasis has long referring clinical provider. Amsel criteria37 had been used been favoured over 7-day-dose treatment, because to test for bacterial vaginosis at the clinics. Symptoms for adherence to treatment is not an issue, especially if the bacterial vaginosis and trichomoniasis are often similar,7 patient is under direct observation by a clinician. Despite and Amsel has low predictability for bacterial vaginosis.38 their willingness to be randomised, 30% of the women 7-day-dose metronidazole is the first-line treatment for thought it would be difficult or somewhat difficult to take bacterial vaginosis7 and is more effective than single-dose the medicine for 7 days. Indeed, in this study self- metronidazole for trichomoniasis, providing further reported adherence was higher among patients receiving rationale for recommending 7-day-dose metronidazole as the single dose compared with patients receiving the first-line treatment for trichomoniasis. 7-day dose, but adherence in both arms was very high. The high rates of positive T vaginalis results at TOC in Providers who have concerns about patient adherence both treatment groups are of concern; 11% of patients will need to consider the benefits of the 7-day dose and receiving the 7-day dose tested positive at TOC, and the convenience of the single dose. 19% of patients receiving the single dose tested positive The study population consisted of non-pregnant women at TOC. These findings suggest that the CDC re-​ with trichomoniasis living mainly in urban areas, with a commendation to rescreen women treated for high rate of co-occurring bacterial vaginosis. The majority trichomoniasis should be upheld even for those women of women who enrolled were African American. Although that receive the 7-day dose. our results might not be generalisable to all women with Although NAAT has greater sensitivity to detect trichomoniasis, they are probably generalisable to most, T vaginalis than culture, it is not clear how long remnant since the study population represents those at high risk T vaginalis DNA stays in vaginal fluids when it is no longer for trichomoniasis.7,32 Our study population was mostly a live parasite. We decided to request TOC visits 4 weeks symptomatic, but most women with trichomoniasis are after completion of treatment because three previous asymptomatic,42 and the effect of asymptomatic studies found 0–15% false positives if the TOC visits were trichomoniasis on reproductive morbidity is not well 3 weeks after completion of treatment.30,31,39 When we known.7 More studies of women with asymptomatic tested for T vaginalis with culture, we found a reduced trichomoniasis are needed. presence of T vaginalis at TOC in both treatment groups, About a third of the cohort had side-effects that were but the relative risk was similar (table 2). This result minimal and did not appear to differ by treatment group. suggests that false positives from NAAT did not have a There were, however, two reported spontaneous abortions major effect on our results. among women receiving the 7-day dose. The difference We chose to use an open-label study design to factor in between groups might be due to chance, and reviews43,44 real use conditions, although masking participants to have found that multiple doses of metronidazole are safe treatment is often preferred. Because of the open-label in pregnancy. Strengths of the study were that study design, treatment duration and follow-up times randomisation appeared to work well, as baseline were different for each treatment group, which could characteristics were similar by group (table 1), loss to have resulted in differences in sexual exposure. However, follow-up was minimal (13%), and the treatment group we did not find any significant differences in sexual was masked from the laboratory technicians when exposure between groups after treatment. Although self- outcome measures were recorded. Moreover, the reported sexual behaviour might not be accurate, we intention-to-treat, modified intention-to-treat, and other used computer-assisted interviews, which have been sensitivity analyses were consistent with each other. shown to reduce social desirability bias.40,41 Furthermore, Combined with our previous work,45,46 this study provides reporting bias should be the same across groups, as all strong evidence that 7-day-dose metronidazole is a better participants received similar counselling. treatment option for women with trichomoniasis than An important limitation of the study was that enrolment single-dose metronidazole, and recommendations should was much lower than planned; maybe our sample size be adapted accordingly. calculation was too conservative. Fewer participants could Contributors result in reduced power for the primary outcomes and less PK, NS, and LM contributed to the data analysis. PK, CAM, LAM, RAL, certainty about the effect measure, particularly for the JRS, LB, SNT, NS, and DHM wrote the Article. CAM, LAM, RAL, JRS, LB, and SNT contributed to the data collection. DHM, PA, WES, MB, analysis stratified by bacterial vaginosis. Our calculations and JMC did the laboratory work and wrote the corresponding sections were based on inputs from our unpublished pilot study of the Article. with HIV-uninfected women, and from our previous Declaration of interests study of trichomoniasis treatment in HIV-infected All authors have received a portion of their salary from the National women.22 It is possible we did not have enough power to Institutes of Health/National Institute of Allergy and Infectious Diseases detect a difference by bacterial vaginosis status, but the (1R01AI097080–01A1), via their institutions. CAM has been a consultant for Lupin Pharmaceuticals. LAM has received consulting fees from relative risks by bacterial vaginosis status did not indicate Gilead Sciences and ViiV Healthcare. LAM and LB have received a trend, so it is unlikely that a larger sample size would research support from Hologic, Becton Dickinson, Gilead Sciences, have led to a different conclusion. and ViiV Healthcare. JRS received research support from Hologic, www.thelancet.com/infection Vol 18 November 2018 1257 Articles

Becton Dickinson, Cepheid, and Symbiomix. SNT has received research 14 Thin RN, Symonds MAE, Booker R. Double-blind comparison of a support from Becton Dickinson, Hologic, Cepheid, Beckman Coulter, single dose and a five-day course of metronidazole in the treatment of Roche, ELITech, GlaxoSmithKline, Melinta, and Entasis. SNT has also trichomoniasis. Br J Vener Dis 1979; 55: 354–56. received consulting, peer educator, and advisory board honoraria from 15 Woodcock KR. Treatment of trichomonal vaginitis with a single oral Hologic and GlaxoSmithKline. DHM has been a consultant for BioFire dose of metronidazole. Br J Vener Dis 1972; 48: 65–68. Diagnostics and GlaxoSmithKline. 16 Schulz KF, Altman DG, Moher D. CONSORT 2010 statement: updated guidelines for reporting parallel group randomised trials. Acknowledgments BMJ 2010; 340: c332. This research was supported by a grant from the National Institutes of 17 Roth AM, Williams JA, Ly R, et al. Changing sexually transmitted Health/National Institute of Allergy and Infectious Diseases infection screening protocol will result in improved case finding for (1R01AI097080–01A1). PA and WES from the CDC provided their trichomonas vaginalis among high-risk female populations. services in kind. We thank the Data Safety and Monitoring Board, Sex Transm Dis 2011; 38: 398–400. including Charlotte Gaydos, David Mushatt, Jeffery Burton, and 18 Hollman D, Coupey SM, Fox AS, Herold BC. Screening for Russell Van Dyke. We thank Caroline Deal and Hagit David from the Trichomonas vaginalis in high-risk adolescent females with a new Division of Microbiology and Infectious Diseases of the National transcription-mediated nucleic acid amplification test (NAAT): Institute of Allergy and Infectious Diseases and DMID for their support. associations with ethnicity, symptoms, and prior and current STIs. At University of Alabama at Birmingham we thank the clinical and J Pediatr Adolesc Gynecol 2010; 23: 312–16. laboratory staff, including Hanna Harbison, Saralyn Richter, 19 Schwebke JR, Hobbs MM, Taylor SN, et al. Molecular testing for Rhonda Whidden, Meghan Whitfield, Christen Press, Jim Alosi, Trichomonas vaginalis in women: results from a prospective U.S. Ann Dillashaw, Charles Rivers, Cheri Aycock, and Keonte Graves. clinical trial. J Clin Microbiol 2011; 49: 4106–11. At University of Mississippi Medical Center we thank Melverta Bender 20 Schwebke JR, Gaydos CA, Davis T, et al. Clinical evaluation of the and Jennifer Brumfield. At Louisiana State University we thank Cepheid Xpert TV assay for detection of Trichomonas vaginalis with Camille Fournet, and at the Louisiana State University Health Sciences prospectively collected specimens from men and women. J Clin Microbiol 2018; 56: e01091–17. Center we thank the laboratory staff, including Catherine Cammarata, 21 Van Der Pol B, Williams JA, Taylor SN, et al. Detection of Judy Burnett, and Denise Diodene. We also thank the data management Trichomonas vaginalis DNA by use of self-obtained vaginal swabs staff at Tulane University, including Lauren Ostrenga and Scott White. with the BD ProbeTec Qx assay on the BD Viper system. The findings and conclusions in this study are those of the authors and J Clin Microbiol 2014; 5: 885–89. do not necessarily represent the views of the National Institutes of 22 Kissinger P, Mena L, Levison J, et al. A randomized treatment trial: Health, National Institute of Allergy and Infectious Diseases, or single versus 7-day dose of metronidazole for the treatment of the CDC. Trichomonas vaginalis among HIV-infected women. References J Acquir Immune Defic Syndr 2010; 55: 565–71. 1 Newman L, Rowley J, Vander Hoorn S, et al. Global estimates of 23 Gatski M, Martin DH, Levison J, et al. The influence of bacterial the prevalence and incidence of four curable sexually transmitted vaginosis on the response to Trichomonas vaginalis treatment among infections in 2012 based on systematic review and global reporting. HIV-infected women. Sex Transm Infect 2011; 87: 205–08. PLoS One 2015; 10: e0143304. 24 Fichorova RN. Impact of T. vaginalis infection on innate immune 2 Patel EU, Gaydos CA, Packman ZR, Quinn TC, Tobian AAR. responses and reproductive outcome. J Reprod Immunol 2009; Prevalence and correlates of Trichomonas vaginalis infection among 83: 185–89. men and women in the United States. Clin Infect Dis 2018; 25 Kissinger P, Schmidt N, Mohammed H, et al. Patient-delivered 67: 211–217. partner treatment for Trichomonas vaginalis infection: a randomized 3 Cherpes TL, Wiesenfeld HC, Melan MA, et al. The associations controlled trial. Sex Transm Dis 2006; 33: 445–50. between pelvic inflammatory disease, Trichomonas vaginalis 26 Nye MB, Schwebke JR, Body BA. Comparison of APTIMA infection, and positive herpes simplex virus type 2 serology. Trichomonas vaginalis transcription-mediated amplification to wet Sex Transm Dis 2006; 33: 747–52. mount microscopy, culture, and polymerase chain reaction for 4 Silver BJ, Guy RJ, Kaldor JM, Jamil MS, Rumbold AR. diagnosis of trichomoniasis in men and women. Trichomonas vaginalis as a cause of perinatal morbidity: Am J Obstet Gynecol 2009; 200: 188 e1–7. a systematic review and meta-analysis. Sex Transm Dis 2014; 27 Biomed Diagnostics, Inc. InPouch TV: instructions for use. July, 2017. 41: 369–76. http://biomeddiagnostics.com/resources/files/100-001%20IFU%20 5 Allsworth JE, Ratner JA, Peipert JF. Trichomoniasis and other InPouch%20TV-Rev_N%20(1).PDF (accessed July 1, 2014). sexually transmitted infections: results from the 2001–2004 National 28 US FDA. Clinical laboratory improvement amendments (CLIA). Health and Nutrition Examination Surveys. Sex Transm Dis 2009; https://www.fda.gov/MedicalDevices/ 36: 738–44. DeviceRegulationandGuidance/IVDRegulatoryAssistance/ 6 Kissinger P, Adamski A. Trichomoniasis and HIV interactions: ucm124105.htm (accessed July 1, 2014). a review. Sex Transm Infect 2013; 89: 426–33. 29 Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing 7 Workowski KA, Bolan GA. Sexually transmitted diseases treatment bacterial vaginosis is improved by a standardized method of Gram guidelines, 2015. MMWR Recomm Rep 2015; 64: 1–137. stain interpretation. J Clin Microbiol 1991; 29: 297–301. 8 WHO. Guidelines for the management of sexually transmitted 30 Van Der Pol B, Williams JA, Orr DP, Batteiger BE, Fortenberry JD. infections. World Health Organization, Feb 1, 2004. http://apps. Prevalence, incidence, natural history, and response to treatment of who.int/iris/bitstream/handle/10665/42782/9241546263_eng.pdf;js Trichomonas vaginalis infection among adolescent women. essionid=125EF38AA4513A9D43CDCC252337A32D?sequence=1 J Infect Dis 2005; 192: 2039–44. (accessed July 1, 2014). 31 Williams JA, Ofner S, Batteiger BE, Fortenberry JD, Van Der Pol B. 9 Johnson G. Tinidazole (Tindamax) for trichomoniasis and bacterial Duration of polymerase chain reaction-detectable DNA after vaginosis. Am Fam Physician 2009; 79: 102–05. treatment of Chlamydia trachomatis, Neisseria gonorrhoeae, and 10 Howe K, Kissinger PJ. Single-dose compared with multidose Trichomonas vaginalis infections in women. Sex Transm Dis 2014; metronidazole for the treatment of trichomoniasis in women: 41: 215–19. a meta-analysis. Sex Transm Dis 2017; 44: 29–34. 32 Kissinger P. Trichomonas vaginalis: a review of epidemiologic, 11 Aubert JM, Sesta HJ. Treatment of vaginal trichomoniasis. clinical and treatment issues. BMC Infect Dis 2015; 15: 307. Single, 2-gram dose of metronidazole as compared with a seven-day 33 Feaster DJ, Mikulich-Gilbertson S, Brincks AM. Modeling site course. J Reprod Med 1982; 27: 743–45. effects in the design and analysis of multi-site trials. 12 Csonka GW. Trichomonal vaginitis treated with one dose of Am J Drug Alcohol Abuse 2011; 37: 383–91. metronidazole. Br J Vener Dis 1971; 47: 456–58. 34 Meites E, Gaydos CA, Hobbs MM, et al. A review of 13 Hager WD, Brown ST, Kraus SJ. Metronidazole for vaginal evidence-based care of symptomatic trichomoniasis and trichomoniasis. Seven-day vs single-dose regimens. JAMA 1980; asymptomatic Trichomonas vaginalis infections. 244: 1219–20. Clin Infect Dis 2015; 61 (suppl 8): S837–48.

1258 www.thelancet.com/infection Vol 18 November 2018 Articles

35 Spear GT, Sikaroodi M, Zariffard MR, Landay AL, French AL, 41 Richman WL, Kiesler S, Weisband S, Drasgow F. A meta-analytic Gillevet PM. Comparison of the diversity of the vaginal microbiota study of social desirability distortion in computer-administered in HIV-infected and HIV-uninfected women with or without questionnaires, traditional questionnaires, and interviews. bacterial vaginosis. J Infect Dis 2008; 198: 1131–40. J Appl Psychol 1999; 84: 754–75. 36 Torrone EA, Morrison CS, Chen PL, et al. Prevalence of sexually 42 Sutton M, Sternberg M, Koumans EH, McQuillan G, Berman S, transmitted infections and bacterial vaginosis among women in Markowitz L. The prevalence of Trichomonas vaginalis infection sub-Saharan Africa: an individual participant data meta-analysis of among reproductive-age women in the United States, 2001–2004. 18 HIV prevention studies. PLoS Med 2018; 15: e1002511. Clin Infect Dis 2007; 45: 1319–26. 37 Amsel R, Totten PA, Spiegel CA, Chen KC, Eschenbach D, 43 Sheehy O, Santos F, Ferreira E, Berard A. The use of metronidazole Holmes KK. Nonspecific vaginitis. Diagnostic criteria and microbial during pregnancy: a review of evidence. Curr Drug Saf 2015; 10: 170–79. and epidemiologic associations. Am J Med 1983; 74: 14–22. 44 Koss CA, Baras DC, Lane SD, et al. Investigation of metronidazole 38 Sha BE, Chen HY, Wang QJ, Zariffard MR, Cohen MH, Spear GT. use during pregnancy and adverse birth outcomes. Utility of Amsel criteria, Nugent score, and quantitative PCR for Antimicrob Agents Chemother 2012; 56: 4800–05. Gardnerella vaginalis, Mycoplasma hominis, and Lactobacillus spp. for 45 Gatski M, Martin DH, Clark RA, Harville E, Schmidt N, diagnosis of bacterial vaginosis in human immunodeficiency Kissinger P. Co-occurrence of Trichomonas vaginalis and bacterial virus-infected women. J Clin Microbiol 2005; 43: 4607–12. vaginosis among HIV-positive women. Sex Transm Dis 2011; 39 Martin D, Burnett J, Taylor S. Trichomonas vaginalis nucleic acid 38: 163–66. clearance following treatment of HIV negative women. 46 Franklin TL, Monif GR. Trichomonas vaginalis and bacterial Sex Transm Infect 2015; 91 (suppl 2): A47 (abstract). vaginosis. Coexistence in vaginal wet mount preparations from 40 Kissinger P, Rice J, Farley T, et al. Application of computer-assisted pregnant women. J Reprod Med 2000; 45: 131–34. interviews to sexual behavior research. Am J Epidemiol 1999; 149: 950–54.

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